The mosquito Aedes aegypti is a primary vector for major arboviruses, and its control is mainly based on the use of insecticides. Caffeine and spent coffee grounds (CG) are potential agents in controlling Ae. aegypti by reducing survival and blocking larval development. In this study, we analyzed the effects of treatment with common CG (CCG: with caffeine), decaffeinated CG (DCG: with low caffeine), and pure caffeine on the survival, behavior, and morphology of the midgut of Ae. aegypti under laboratory conditions. Third instar larvae (L3) were exposed to different concentrations of CCG, DCG, and caffeine. All compounds significantly affected larval survival, and sublethal concentrations reduced larval locomotor activity, delayed development, and reduced adult life span. Damage to the midgut of treated larvae included changes in epithelial morphology, increased number of peroxidase-positive cells (more abundant in DCG-treated larvae), and caspase 3-positive cells (more abundant in CCG-treated larvae), suggesting that the treatments triggered cell damage, leading to activation of cell death. In addition, the treatments reduced the FMRFamide-positive enteroendocrine cells and dividing cells compared to the control. CG and caffeine have larvicidal effects on Ae. aegypti that warrant field testing for their potential to control mosquitoes.

Despite the increased attention for temporal aspects of stressor interactions and for effects of warming in ecotoxicological studies, we lack knowledge on how different exposure durations to warming may affect pesticide sensitivity. We tested how three types of exposure duration to 4 °C warming (acute, developmental and transgenerational exposure to 24 °C vs 20 °C) shape the effect of the pesticide chlorpyrifos on two ecologically relevant fitness-related traits of mosquito larvae: heat tolerance and antipredator behaviour. Transgenerational (from the parental generation) and developmental (from the egg stage) warming appeared energetically more stressful than acute warming (from the final instar), because (i) only the latter resulted in an adaptive increase of heat tolerance, and (ii) especially developmental and transgenerational warming reduced the diving responsiveness and diving time. Exposure to chlorpyrifos decreased the heat tolerance, diving responsiveness and diving time. The impact of chlorpyrifos was lower at 24 °C than at 20 °C indicating that the expected increase in toxicity at 24 °C was overruled by the observed increase in pesticide degradation. Notably, although our results suggest that transgenerational warming was energetically more stressful, it did reduce the chlorpyrifos-induced negative effects at 24 °C on heat tolerance and the alarm escape response compared to acute warming. Our results provide important evidence that the exposure duration to warming may determine the impact of a pesticide under warming, thereby identifying a novel temporal aspect of stressor interactions in risk assessment.

Due to increased use of agrochemicals and growing concerns about ecotoxicology, the development of new insecticides, moving away from those with neurotoxic and broad spectrum effects towards insecticides that are safer for the environment and nontarget beneficial species, has been a research priority. Novaluron stands out among these newer insecticides, is an insect growth regulator that is used for the control of insect pests in crops grown close to mulberry plantations. Mulberry serves as food for the silkworm Bombyx mori, which is a nontarget insect of great economic importance to silk production. We investigated the lethal and sublethal effects of Novaluron on the development of B. mori. Larvae were segregated into experimental groups: the control groups (CGs) and the treatment groups (TGs), which were treated with the Novaluron concentration of 0.15 mL/L. Following exposure, we analyzed: larval mortality, changes in the insect life cicle and cytotoxic effects on the midgut cells. This is the first report about the Novaluron’s effects on B.mori. We detected rupture in the integument, complete cessation of feeding, late development, incomplete ecdysis and production of defective cocoons. After 240 h of exposure, there was 100% mortality in TG larvae exposed in the 3rd instar and 20% mortality from larvae exposed in the 5th instar. Cytotoxic effects was observed, such as dilation of cells, emission of cytoplasmic protrusions, extreme rarefaction of the cytoplasm and nuclei, dilation of the endoplasmic reticulum in addition to changes in mitochondria, the presence of large digestive vacuoles and intercellular spaces and the presence of active caspase. Novaluron exposure impairs the midgut and may affect the physiological functions of this organ. Novaluron additionally compromises several phases of insect development, indicating the importance of toxicology studies that utilize different life stages of nontarget species to evaluate the safe use of insecticides.

The hydrocarbon phenanthrene is an organic compound commonly found in the environment. In aquatic ecosystems, it is highly toxic to organisms, although little is known about its effects on sediment-dwelling organisms. The purpose of this study was to evaluate phenanthrene effects on biochemical, histological, and ontogenetic levels in larvae of the sediment-dwelling invertebrate Chironomus sancticaroli at acute and chronic exposure. Lethal concentrations were estimated and toxicity (acute-96 h and chronic- 8 d) tests were performed at phenanthrene concentrations from 0.12 to 1.2 mg L⁻¹. At acute and chronic exposure, we evaluated acetylcholinesterase (AChE), alpha esterase (EST-α), and beta esterase (EST-β) activities as well as histological alterations. In the assays with chronic exposure, effects on larval development were estimated using antennae length (instar estimative) and body length (growth estimative). The EST-α showed a significantly increased activity after 48 h at acute exposure to high concentrations of phenanthrene, while EST-β activity was increased after 48 and 72 h at acute exposure at higher concentrations and at 0.12 mg L⁻¹ at chronic exposure. At acute exposure, the midgut showed alterations such as brush border disruption, gastric caeca regression, and lumen area reduction; the fat body showed nuclear alteration in the trophocytes, while the Malpighian tubules showed brush border reduction and the salivary glands were subject to cytoplasm vacuolation. At chronic exposure, the same alterations were observed, in addition to vacuolar coalescence in the trophocytes of the fat body. Regarding larval development, a reduction of body length was observed with increasing phenanthrene concentrations. Similarly, molting was delayed; in the control group, all larvae were in the fourth instar, while at higher phenanthrene concentrations, larvae were predominantly in the third instar. Phenanthrene had toxic effects on this chironomid, indicating risks for natural populations.

Lange's metalmark butterfly, Apodemia mormo langei Comstock, is in danger of extinction due to loss of habitat caused by invasive exotic plants which are eliminating its food, naked stem buckwheat. Herbicides are being used to remove invasive weeds from the dunes; however, little is known about the potential effects of herbicides on butterflies. To address this concern we evaluated potential toxic effects of three herbicides on Behr's metalmark, a close relative of Lange's metalmark. First instars were exposed to recommended field rates of triclopyr, sethoxydim, and imazapyr. Life history parameters were recorded after exposure. These herbicides reduced the number of adults that emerged from pupation (24–36%). Each herbicide has a different mode of action. Therefore, we speculate that effects are due to inert ingredients or indirect effects on food plant quality. If these herbicides act the same in A. mormo langei, they may contribute to the decline of this species.

Morphological tools can assist in the evaluation of effects of insecticides on non-target insects. Pyriproxyfen, a juvenile hormone analog, is known to interfere with growth and metamorphosis of insects. However, there are studies showing indirect effects on natural enemies, including green lacewings. Few prior studies describe morphological effects of pyriproxyfen on target insect organs, especially on natural enemies. Through morphological tools, this study aimed to characterize the midgut and fat body, both important organs of digestion and great metabolic activity respectively, of the predator Ceraeochrysa claveri after chronic exposure to pyriproxyfen. Larvae of C. claveri were fed Diatraea saccharalis egg clusters treated with pyriproxyfen in solution of 50 or 100 mg a.i. L⁻¹ throughout the larval stage. The biological data revealed significant increases in development time, especially in the third instar, and in cumulative mortality from the prepupal into the pupal stage. Morphological analysis of adult midgut (≤24 h old) showed damage including formation of epithelial folds, intercellular spaces, emission of cytoplasmic protrusions. Both fat body regions presented decrease of lipid droplets, vacuolization of trophocytes and mitochondrial injury featuring a multisystemic action. In both organs, pyriproxyfen exposure induced significant oxidative stress by mitochondrial superoxide production. Cytoprotective responses were induced in midgut and fat body cells by augmenting the number of cytoplasmic granules containing calcium and expression of HSP 90. Both organs proved to be efficient in presenting histopathological alterations, showing the sensitivity and applicability of this morphological tool for evaluating other insecticides in non-target organisms.

The decreasing ocean pH seems to adversely affect marine organisms, including crustaceans, which leads to potential threats to seafood safety. The present investigation evaluated the effect of seawater acidification on the edible marine mud crab Scylla serrata instars. The experimental setup was designed using a multi-cell cage based system assembled with 20 pre holed PVC pipes containing 20 individual crabs to avoid cannibalism. The crab instars were exposed to CO₂ driven acidified seawater at pH 7.8 (IPCC forecast pH at the end of the 21ˢᵗ century), 7.6, 7.4, 7.2, and 7.0 for 60 days. The crabs reared in seawater without acidification at pH 8.2 served as control. The present study revealed a notable decrease in survival, feed intake, growth, molting, tissue biochemical constituents, minerals, chitin, and alkaline phosphatase in S. serrata instar reared in acidified seawater, denotes the adverse effect of seawater acidification on crabs. The significant elevations in antioxidants, lipid peroxidation, and metabolic enzymes in all acidified seawater compared to ambient pH indicates the physiological stress of the crabs' instars. The changes in the metabolic enzymes reveal the metabolism of protein and glucose for additional energy required by the crabs to tolerate the acidic stress. Hence, the present study provides insight into the seawater acidification can adversely affect the crab S. serrata.

Species of the genus Microcystis are among the most notorious cyanobacteria in eutrophic lakes worldwide, with ability present adverse effects on many aquatic organisms. In the surface sediments, Microcystis can be ingested by benthic macroinvertebrates such as Chironomus. However, the potential negative effects of Microcystis on Chironomus life history traits remain unclear. In the present study, we investigated the effect of different Microcystis diets on specific behaviors (burrowing activity, locomotion ability) and life history traits of Chironomus pallidivittatus (Diptera, Chironomidae). We also studied the interactive effects of microcystin-producing M. aeruginosa and temperature (15, 20, and 25 °C) stress on chironomid larvae. The results showed that the inhibitory effect on the cumulative emergence and burrowing activity of larvae was more severe when they were fed M. aeruginosa among the three Microcystis diets groups. Locomotion ability (i.e., locomotor distance and velocity) and adult dry weight decreased significantly in the group fed M. aeruginosa. Locomotion was significantly inhibited and mortality increased when the larvae were fed a mixture of M. aeruginosa and M. wesenbergii, which may have been the result of additive or synergistic effect of the toxins. Under the stress of lower temperature, C. pallidivittatus larvae exhibited weaker locomotion and growth ability, and the emerging adults were mostly male. At both the lower and higher temperature conditions, M. aeruginosa cause cumulative emergence decreased, and sex ratio imbalance, which inhibited the reproduction of larvae from the population perspective. The fourth-instar larvae showed better adaption to Microcystis than did the other instars. This study thus highlights the adverse effects of microcystin-producing M. aeruginosa on Chironomus. It also provides a novel perspective on how environmental factors may influence the behavior and life history traits of chironomid larvae, and how they may respond to cyanobacterial blooms and global warming.

Vinclozolin (Vz) is a pollutant found in aquatic environments whose antiandrogenic effects in reproduction are well known in mammals. Although its reproductive effects have been less studied in invertebrates, other effects, including genotoxicity, have been described. Therefore, in this work, we studied the genotoxic effects of Vz in the freshwater benthic invertebrate Chironomus riparius. DNA damage was evaluated with the comet assay (tail area, olive moment, tail moment and % DNA in tail), and the transcriptional levels of different genes involved in DNA repair (ATM, NLK and XRCC1) and apoptosis (DECAY) were measured by RT-PCR. Fourth instar larvae of C. riparius, were exposed to Vz for 24 h at 20 and 200 μg/L. The Vz exposures affected the DNA integrity in this organism, since a dose-response relationship occurred, with DNA strand breaks significantly increased with increased dose for tail area, olive moment and tail moment parameters. Additionally, the lower concentration of Vz produced a significant induction of the transcripts of three genes under study (ATM, NLK and XRCC1) showing the activation of the cellular repair mechanism. In contrast, the expression of these genes with the highest concentration were downregulated, indicating failure of the cellular repair mechanism, which would explain the higher DNA damage. These data report for the first time the alterations of Vz on gene transcription of an insect and confirm the potential genotoxicity of this compound on freshwater invertebrates.

Using Artemia salina (A. salina) cysts (capsulated and decapsulated) and larvae [instar I (0–24 h), II (24–48 h) and III (48–72 h)] as experimental models, developmental toxicity of oxidized multi-walled carbon nanotubes (O-MWCNTs) was evaluated. Results revealed that hatchability of capsulated and decapsulated cysts was significantly decreased (p < 0.01) following exposure to 600 mg/L for 36 h. Mortality rates were 33.8, 55.7 and 40.7% for instar I, II and III larvae in 600 mg/L. The EC50 values for swimming inhibition of instar I, II and III were 535, 385 and 472 mg/L, respectively. Instar II showed the greatest sensitivity to O-MWCNTs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Effects on hatchability, mortality and swimming were accounted for O-MWCNTs rather than metal catalyst impurities. Body length was decreased with the concentrations increased from 0 to 600 mg/L. O-MWCNTs attached onto the cysts, gill and body surface, resulting in irreversible damages. Reactive oxygen species, malondialdehyde content, total antioxidant capacity and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities were increased following exposure, indicating that the effects were related to oxidative stress. O-MWCNTs were ingested and distributed in phagocyte, lipid vesicle and intestine. Most of the accumulated O-MWCNTs were excreted by A. salina at 72 h, but some still remained in the organism. Data of uptake kinetics showed that O-MWCNTs contents in A. salina were gradually increased from 1 to 48 h and followed by rapidly decreased from 48 to 72 h with a range from 5.5 to 28.1 mg/g. These results so far indicate that O-MWCNTs have the potential to affect aquatic organisms when released into the marine ecosystems.